Studies on the promoters of vertebrate ribosomal RNA genes (-170 to +15) indicate that they have nearly identical functional elements with different nucleic acid sequences and species specific trans-acting elements. The holopromoter of mammalian ribosomal genes has two components: a core promoter element (CPE) and an upstream promoter element (UPE). The core promoter element is necessary and sufficient to direct accurate transcription. However, experimental evidence suggests that stable transcription initiation complexes form more tightly or more rapidly on the holopromoter. Deletion mutants have been used to map the upstream promoter element of rat rDNA between nucleotides -114 and -167 (+1 being the transcription initiation site). We propose to define the specific nucleotides of the UPE required to affect stable complex formation, and to determine the mechanism by which the UPE and CPE interact. In vitro order-of-addition transcription experiments, and in vivo expression assays will be used to determine the mechanism of action of the UPE. We have partially purified one of the trans-acting factors required for species specific ribosomal gene expression. This factor will be purified to homogeneity and its role in transcription identified. Other fractions, required for transcription and/or elevated levels of gene expression have been identified, and they too will be purified and their mechanisms of action defined using DNA-binding assays, DNA footprinting, nuclease sensitivity and in vitro transcription experiments. One fraction that stimulates transcription has been identified, and is being isolated and characterized. The objective of this proposal is the definition of the cis-acting elements and trans-acting factors required for ribosomal RNA synthesis. In addition, complementation assays combining extracts and/or fractions from tumor and normal cells, will be carried out to elucidate the factor(s) responsible for the elevated levels of rRNA synthesis in tumor cells. The long-term objective of this research is to determine the mechanism(s) by which ribosomal gene transcription is regulated. These experiments will increase our understanding of the regulation of ribosomal gene expression and the mechanism of action of eukaryotic promoters in general.